Intracellular signaling pathways depend upon appropriate and unique subcellular locations of their constituent proteins. Mechanisms responsible for reversibly targeting peripheral membrane proteins to different cellular membranes are poorly understood. This research grant will address this question in the context of heterotrimeric (alpha beta gamma) G proteins. G proteins act as molecular switches to relay information from activated receptors to appropriate effector proteins (e.g. adenylyl cyclase, phospholipase C1 cGMP phosphodiesterase, and ion channels). Molecular mechanisms underlying the GTPase cycle of G proteins and its regulation by receptors and effectors are becoming increasingly well understood. Much less well understood are mechanisms responsible for targeting G proteins to their appropriate cellular location, and how a unique cellular environment -and proteins that inteact with G proteins to target or retain them there-affect a G protein's function. The major objectives of this proposal are to elucidate mechanisms of cellular palmitoylation, covalent attachment to cysteines of a 16 carbon fatty acid, of G protein alpha subunits (Galpha) and mechanisms of specific targeting of G alpha to Golgi membrane. The role of G protein betagamma subunits in the palmitoylation of Galpha will be tested by employing strategies-expression of a betagamma-binding protein, (beta-ARKct), and identifying mutations in Galpha that block its binding to betagamma- to disrupt the alpha-betagamma interaction in vivo. The subcellular site of palmitoylation of Galpha will be addressed by examining the palmitoylation of differentially localized Galpha; alpha12 resides at the plasma membrane, whereas alpha13 and a splice varent of alpha12 (salpha12) are found at Golgi membranes. Inhibitors of membrane vesicle transport will be used to further define the cellular pathway for palmitoylation. To understand mechanisms of specific Golgi localizaton fo Galpha, the role of lipid modificatons, both myristoylation and palmitoylation, and betagamma interaction will be tested by expression of mutant Galpha. Amino acids (i.e., structural determinants) in alpha13 and salpha12 that specifiy Golgi localization will be defined by expression and immunofluorescence microscopy localization of mutant and chimeric Galpha. Finally, proteins that interact with and target alpha13 and salpha12 to Golgi membranes will be identified by affinity isolation approaches and yeast two-hybrid screening.